Following return from the Gulf War (GW), Veterans have exhibited of a constellation of symptoms - designated Gulf War Illness (GWI) - that cannot be associated with a single disease. GW Veterans also show increased rates of developing psychological symptoms and psychiatric disorders, along with alterations in hypothalamic-pituitary-adrenal (HPA) axis function and neuroanatomical changes. The precise cause for these symptoms remains unknown. The acetylcholinesterase (AChE) inhibitor pyridostigmine bromide (PB) was used as prophylaxis against the deleterious effects of nerve agents during the GW. When combined with the operational stress experienced by soldiers, this PB exposure has been proposed as one of the causes of the late cognitive dysfunction in GWI. Other studies determined that PB is a causative factor in the development of impaired immune cell function in GW Veterans, which may contribute to the memory deficits observed in GWI. Since vagal cholinergic afferents and efferents control a reflex circuit that regulates inflammation, we propose to investigate the hypothesis that the combined effects of stress and PB exposure result in altered immune function, which then leads to modifications in cholinergic responses in key brain areas that lead to cognitive deficits. Using a preclinical model of combined repeated psychological stress and PB exposure, we will test our overarching hypothesis that the neurocognitive deficits in GWI are related to combined effects of PB and repeated stress on immune function that alters acetylcholine function in hippocampus. This hypothesis will be tested in the following Aims: Aim 1 will examine whether the combination of PB + repeated stress induces HPA axis dysfunction, increases cytokine levels, reduces the activity of cholinergic projections to the hippocampus and modulates vagal medullary centers. Aim 2 will examine hippocampal synaptic re-organization, neuronal and microglia dendritic architecture and neuronal degeneration, analyses which would provide a functional anatomical basis for cognitive deficits observed in RRS + PB rats. Aim 3 will directly assess the combined effects of PB and stress exposure on acetylcholine release in the hippocampus. In vivo microdialysis will be used to examine basal and behaviorally-induced changes in hippocampal acetylcholine levels during the performance of a hippocampal-dependent task and during an exposure to an acute stressor. Aim 4 will more directly assess how stress and PB affect immune function via immunological analyses performed in plasma, spleen, and lymph nodes, as well as microglia isolated from the hippocampus, after exposure to PB +/- repeated stress at early and delayed time points. Successful completion of these studies will demonstrate that the combination of PB + repeated stress exposure elicits peripherally-mediated changes in pro-inflammatory cytokines/chemokines that are mechanistically responsible for deficits in hippocampal cholinergic activity, thereby providing a neurochemical and anatomical basis for behavioral deficits following exposure to PB and stressful events. Most importantly, successful completion of the proposed studies will identify loci for therapeutic intervention that can be quickly implemented for the treatment of GWI in our Veterans.